The cAMP-dependent protein kinase (PKA) is the canonical effector of the β-adrenergic (β-AR)/cAMP cascade and a major physiological regulator of cardiac function. In the adult heart, PKA mediates the cardiotonic effects of acute β-AR stimulation, but persistent PKA activation has been linked to maladaptive remodeling, suggesting that PKA could be an interesting target in heart failure (HF). In the inactive state, PKA is a heterotetramer composed of two regulatory (R) subunits which bind and inhibit two catalytic (C) subunits. Upon β-AR stimulation, cAMP binds to the R subunits, causing the activation of the C subunits. Two types of PKA, type I and type II, vary according to their regulatory subunits, RIα and RIIα, respectively. While type II PKA has been involved in the regulation of cardiac contraction, the role of type I PKA in the heart is less well understood. Indeed, in mice global and cardiomyocyte-specific RIα knockout increase PKA activity and is embryonic lethal due to cardiac development failure. In humans, heterozygous inactivating mutations in the PRKAR1A gene resulting in RIα haploinsufficiency cause Carney Complex (CNC), a rare endocrine disease associated with cardiac myxomas that cause a high morbidity/mortality. Cardiac myxomas are benign, slowly proliferating lesions of sub-endocardial origin but the cells composing these tumours are not clearly identified. CNC-derived cardiac myxoma have been associated with congenital heart defects, suggesting their developmental origin. Based on this information, the two main objectives of this project are i) to determine the consequences of RIα inactivation in the adult mouse heart and in human engineered cardiac tissue and ii) to decipher the precise requirement of type I PKA in early cardiac morphogenesis and myxomagenesis. For this, the partners of this project developed new Cre/Lox mouse models with cardiac cell type specific and temporal knockout of RIα. They will use a unique collection of cardiac myxoma from CNC patients and single cell nuclei RNA sequencing to identify the cell population(s) constituting these tumours. In order to model CNC and explore the impact of RIα haploinsufficiency in human cardiogenesis and myxomagenesis, they will reprogram peripheral blood mononuclear cells from patients carrying an inactivating PRKAR1A mutation into human induced pluripotent stem cells (hiPSC) and differentiate them into different cardiac lineages including cardiomyocytes (hiPSC-CM), neural crest and vascular smooth muscle cells. In addtition, hiPSC-CM cells will be used to produce engineered human heart tissue (EHT) in collaboration with Prof. T. Eschenhagen in Hamburg (Germany). Preliminary results indicate that cardiomyocyte-specific RIα invalidation in adult mice induces chronic PKA activation and results in progressive HF, whereas deletion in cardiac neural crest cells causes partial lethality at embryonic day 15.5 associated with arterial trunk and septal defects reminiscent of congenital heart diseases. The planned work program will further characterize these phenotypes by a combination of state-of-the art techniques including unbiased transcriptomics to determine how RIα controls progenitor cell deployment during embryogenesis and identify new genes involved in maladaptive remodeling in the adult heart that will be validated by in vitro assays and in failing human hearts. The identification of cells composing human cardiac myxoma will pave the way to understand the mechanisms of myxomagenesis in humans and might help to envision therapeutic strategies to prevent the growth of these tumours. Using hiPSC-CM cells to generate EHT will overcome some limitations of hiPSC-CM and provide the first insights into the function of type I PKA in a human cardiac muscle context. Altogether, these findings will allow to better understand the mechanisms by which chronic PKA activation may precipitate congenital heart disease, cardiac myxoma and HF.